ZHX2 Suppresses The Development Of NAFLD And HCC By Transcriptional Repression Of Lipoprotein Lipase

Hepatocellular carcinoma (HCC) is the most frequent primary neoplasm of the liver and is the sixth most common malignancy worldwide. It is also the third leading cause of cancer-related deaths. Most cases of HCC develop on a pre-existing chronic liver disease, usually due to hepatitis C virus (HCV), hepatitis B virus (HBV), or alcohol. However, between 15% and 50% of HCC develops in the absence of a known etiology of liver disease, and different lines of evidence identify in non-alcoholic fatty liver disease(NAFLD) a possible independent risk factor for occurrence of HCC. Given the high incidence of NAFLD and its close correlation with HCC, it is urgent to reveal the possible mechanism underlying the pathogenesis of NAFLD progressing to HCC, which may help to provide newly effective targets for the prevention and treatment of NAFLD-related HCC.The pathogenesis of NAFLD is complicated and the mechanisms linking NAFLD pathogenesis to HCC development is not clear. Abnormal lipid metabolism leading to increased free fatty acid (FFA) and hepatocyte lipid deposition is the prerequisite for the development of NAFLD. More and more evidence showed disorder of lipid metabolism in many solid tumors, including increased de novo FA synthesis, lipolysis, and FFA uptake. Multiple oncogenes and tumor suppressor genes were found to be involved in regulating the metabolic pathway in tumors. Therefore, metabolism disorder mediated by oncogenes and tumor suppressor genes could be a critical mechanism of which NAFLD occur and develop into HCC.The Zinc-Fingers and Homeoboxes 2 (ZHX2) gene is a member of a small gene family that also includes ZHX1 and ZHX3. Two-hybrid studies indicate that ZHX2 can form homodimers as well as heterodimers with other ZHX family members and with NF-YA via HD1 domain to act as a transcriptional repressor. Consistent with these data, ZHX2 represses expression of liver cancer biomarkers, such as alpha-fetoprotein (AFP) and glypican-3 (GPC3). We recently demonstrated that ZHX2 down-regulate the cyclin A and cyclin E expression in HCC cell lines and identified ZHX2 as a new tumor suppressor gene in HCC.Genetic analysis of combined hyperlipidemia and atherosclerosis showed that ZHX2 was a susceptibility gene of hyperlipidemia. Meanwhile, liver-specific transgenic expression of ZHX2 in BALB/cJ mice increases plasma triglycerides. All of the above indicates that there is a relationship between ZHX2 and lipid metabolism disorders. However, the role of ZHX2 regulating lipid metabolism in related liver diseases has not been clear.Here we aim to explore the roles and mechanisms of ZHX2 in the development of NAFLD and HCC, and to study the function of ZHX2 in improving multiple drug resistance of tumor. Part Ⅰ ZHX2 suppresses the development of NAFLD and HCC by transcriptional repression of lipoprotein lipaseThe process of lipid metabolism is complex, including fatty acid uptake, lipoprotein lipolysis and trafficking, fatty acid de novo synthesis, fatty acid β-oxidation and ketogenesis. Related studies indicated that ZHX2 was related to plasma lipid metabolism disorders. Our preliminary gene expression profile chip data suggested that ZHX2 expression had correlation with a series of lipid metabolism related gene expression, and lipoprotein lipase (LPL) is one of them. LPL is a key enzyme that hydrolyzes the TGs in chylomicrons (CM) or very low-density lipoproteins (VLDL). LPL has also been found to have an additional non-catalytic "bridging" function, allowing for the accumulation and cellular uptake of lipoproteins via receptor-mediated endocytosis. With an increasing number of studies, LPL is highly expressed in breast and liposarcoma tumors and has been shown to be a marker of poor prognosis in these solid tumors, indicating that LPL involves in the tumorigenesis. However, it is not completely known whether LPL is involved in lipid metabolism regulated by ZHX2 or in the inhibition of HCC development mediated by ZHX2.Here, in this study, we aim to describe the roles and mechanisms of ZHX2 involved in NAFLD and HCC via influencing lipid metabolism by regulating LPL. I.ZHX2 significantly suppresses the expression of LPL in various cell linesIn order to clarify the regulation of LPL by ZHX2 in vitro, pcDNA3.0-ZHX2-HA was transfected in tumor cell lines (HepG2,BEL7402 and HEK293) with low endogenous ZHX2, and ZHX2 siRNAs were transfected in tumor cell lines (SMMC7721 and QSG7701) with high endogenous ZHX2.The expression of ZHX2 and LPL by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western Blot(WB). Results showed that ZHX2 overexpression down-regulated the expression of LPL, meanwhile LPL expression was upregulated by interfereing with ZHX2. ALL above results suggests that ZHX2 negatively regulates the expression of LPL. II.ZHX2 negatively regulates the activity of LPL promoter via Octamer domain1. ZHX2 significantly suppresses the activity of LPL promoter.First, we constructed the promoter reporter plasmid of LPL (-1678-+67), named as pGL3-LPLp. In order to detect the effects of ZHX2 on LPL promoter activity, report plasmid pGL3-LPLp was cotransfected with pcZHX2 in HepG2 and CHO, and with ZHX2 siRNAs in SMMC7721 and QSG7701 respectively. ZHX2 overexpression could significantly inhibit the activity of LPL promoter was identified by luciferase reporter assays. Meanwhile the LPL activity increased when ZHX2 was knocked down.2. ZHX2 regulates LPL promoter through-96--38nt region. In order to find the key domain by which ZHX2 regulates LPL promoter, we analysed the transcriptional factor binding sites of LPL promoter by TFSEARCH. According to the analysis, we constructed a series of promoter reporter plasmid of LPL (upstream:-981,-816,-666,-430,-196,-96,-38 and downstream:+67). Results of cotransfection assays showed that LPL promoter containing the fragment from-38 to+67 was not affected by ZHX2.3. ZHX2 regulates the activity of LPL promoter via Octamer.According to the distribution of transcriptional factor binding sites, we constructed LPL promoter containing the fragment from-52 to+67. Results of cotransfection assays showed that ZHX2 could still repress its activity. There is only one important domain called Octamer among the region-52～-8. In order to identify the importance of Octamer, we mutated this domain in the pGL3-LPLp-96 plasmid, and getted the pGL3-LPLp-96-Octamer-mutant. The results of cotransfection and luciferase reporter assays showed that ZHX2 could not repress the activity of the mutant plasmid.To further verify the conclusion above, the wild type and mutant Octamer from fragment-46～-39 of LPL promoter was cloned into pGL3-promoter to prepare the promoter reporter plasmids of octamer, named as pGL3-Octamer-promoter and pGL3-Octamer-mutant-promoter. Consistently, ZHX2 could not surpress the activity of the mutant plasmid.One other thing to note here is that there is a NF-YA binding site in the region-70--65, which is near to Octamer in the LPL promoter. It has been clarified that NF-YA is an important transcriptional factor for LPL promoter and ZHX2 can combine with NF-YA to repress a serious of target genes. In order to identify whether NF-YA plays a role in the regulation of LPL by ZHX2, we cotransfected the NF-YA siRNA and pcDNA3.0-ZHX2-HA with LPL promoter. The results suggested that both NF-YA siRNA and pcDNA3.0-ZHX2-HA could influence the activity of LPL promoter separately, but never interfered with each other.4. ZHX2 interacts with Octamer domain of LPL promoter, confirmed by EMSA and ChIP assay.HepG2 cells were transfected with pcDNA3.0-ZHX2-HA.48h after transfection, cells were collected and sonicated until the DNA was sheared to an average size of 200-1000bp. Supernatants obtained after centrifugation were used for immunoprecipitations using anti-HA antibody or control IgG. DNA purified from the immunoprecipitates was used for PCR amplification. Results showed that ZHX2 could bind with the-96～+67 region of LPL promoter.HEK293 cells were transfected with pcDNA3.0-ZHX2-HA or pcDNA3.0 respectively, and then extracted the nuclear protein. The result of electrophoretic mobility shift assay showed that, ZHX2 nuclear protein could bind to the Octamer probe. Additionally, the supershift line confirmed that ZHX2 could specifically bind to Octamer domain.5. ZHX2 can combine with Oct-1 confirmed by Co-IP.ZHX2 has HD1 function domain which can interact with protein. HepG2 cells were transfected with pcDNA3.0-ZHX2-HA and then extracted the total protein. The result of immunoprecipitation showed that Oct-1 protein could bind to ZHX2. III.ZHX2 alleviates the steatosis in vitro and in vivo by suppressing the expression of LPL LPL is a key enzyme of lipolysis, and mediates the lipoprotein uptake of liver. Therefore, LPL plays important roles in lipid metabolism related diseases. The regulation of LPL by ZHX2 suggests that ZHX2 involves in lipid metabolism related diseases. To test this hypothesis, we designed in vitro and in vivo studies as following: 1. ZHX2 inhibits the lipid accumulation in HepG2 cells inducing by fat emulsion (1) ZHX2 expression is negative correlated with LPL expression in NAFLD cell model induced by fat emulsion.HepG2 cells were cultured with different concentrations of fat emulsion (0.2%,0.4%,0.8%,1%,2%). And we finally chose 0.4% fat emulsion to induce lipid deposit, as this concentration would not injured cells directly. Steatosis of HepG2 cells was induced by different concentrations (0.2%,0.4%,1%) fat emulsion. We found that ZHX2 expression decreased and LPL expression increased as steatosis degree aggravating.(2) ZHX2 inhibits the lipid accumulation in HepG2 cells inducing by fat emulsion and overexpression of LPL can reverse this inhibition partly.pEGFP-ZHX2 was transfected into HepG2 cell.24 hours later, added 0.4% fat emulsion. Another 24 hours later, HepG2 cells were stained by oil-red O and DAPI. The results acquired from fluorescence microscope showed that EGFP-ZHX2 positive cells had a minor steatosis in comparison to other cells.In order to clarify the role of LPL in the process which ZHX2 inhibits steatosis of hepatocytes, we constructed LPL overexpression vector, called pcDNA3.0-LPL-HA. pcZHX2 combination with different doses of pcLPL were co-transfected into HepG2 cells, and then added 0.4% fat emulsion to induced the steatosis.48 hours later, the content of TG in cell lysate was detected. The results confirmed that LPL could promote the lipid deposit of HepG2 cell, and LPL could reverse the inhibition function of ZHX2 in lipid accumulation.2. ZHX2 inhibits the development of mouse NAFLD by negatively regulating the LPL expression(1) The expression of ZHX2 in NAFLD mouse model.To further clarifying the role of ZHX2 and LPL in NAFLD, two types of mouse NAFLD model induced by Methionine-Choline-Deficient (MCD) and High-fat diet (HFD) were introduced. The expressions of ZHX2 and LPL in mouse liver were detected by RT-PCR. The fact that ZHX2 expressing level was diminished in NAFLD mouse liver suggested that ZHX2 involved in the pathogenesis of NAFLD by regulating LPL.(2) The degree of NAFLD aggravates when ZHX2 is interfered.C57BL/6 mice were administrated lenti-virus-Afrl-shRNA or lenti-virus-negative control by tail vein high pressure injection. One week later, MCD diet was started. During the experiment, body weight was measured once a week. Two weeks later, the liver was removed, weighed, and cut into small pieces. Liver histopathology was observed, and the contents of triglycerides in liver homogenates were evaluated. For other histological analyses, 10-p.m cryosections were prepared. Some cryosections were stained with Oil Red O to evaluate hepatic fat deposits.3-μm paraffin sections were made for ordinary hematoxylin and eosin (H&E) staining. The results indicated that, the NAFLD model was successfully induced. When ZHX2 was interfered, the degree of NAFLD aggravated.(3) ZHX2 inhibits the steatosis of mouse liver, but overexpression of LPL can reverse this inhibition partially.To further identify the role of ZHX2 involved in pathogenesis by regulating LPL, ZHX2 and LPL expression vectors were administrated by tail vein high pressure injection. Two days later, MCD diet was started. Other treatments were the same as above. The results showed that, LPL exacerbated NAFLD while ZHX2 protected liver from NAFLD, and LPL could reverse the function of ZHX2 in the pathogenesis of fatty liver.IV.ZHX2 represses the growth of HCC by down-regulating the expression of LPLIt was reported that the expression of LPL increased in breast cancer and liposarcoma, and LPL promoted breast cancer cells growth. In order to study the role of LPL in the regulation of HCC cell growth by ZHX2, we designed the experiments in vitro and in vivo:1. The expression of LPL in HCC samples is higher than in paratumor samples and ZHX2 expression is negative correlated with LPL expression.We collected 22 HCC clinical samples to detect the expression of LPL by Real-time quantitative PCR (Q-PCR). The results showed that the expression of LPL in cancer tissues was significant ly higher than that in paratumor tissues (p<0.05).Then the results acquired by immunohistochemistry in HCC tissue chip showed that the expression of LPL in cancer tissues was significant ly higher than that in paratumor tissues (p<0.05). We calculated the expression of ZHX2 and LPL by way of serial section in 97 HCC. We found that ZHX2 expression was negative correlated to LPLexpression (p<0.05).2. LPL stimulates HepG2 cell growth in a lipid-dependent manner.HepG2 cancer cells were tranfected with pcDNA3.0-LPL-HA or pcDNA3.0. Then cells were grown for 72 h in media containing 10% complete fetal calf serum or in media containing 1% BSA or in media containing 1% BSA plus the 0.1% fat emulsion. The results indicated that LPL could promote the growth of HepG2 cells when media containing sufficient lipoprotein, such as 10% FCS.The growth of HepG2 cells were not influenced when the media was deficient with any lipid, such as 1%BSA. But when add fat emusion to the 1%BSA, the function of LPL was recovered.3. LPL reverses the growth inhibition of HepG2 by ZHX2 depending on the presence of lipid.HepG2 cells were transfected with plasmids:pcDNA3.0, pcDNA3.0-ZHX2-HA, pcDNA3.0-LPL-HA, ZHX2 plus LPL. And then these cells were grown x72 h in media containing 10% complete fetal calf serum or in media containing 1% BSA or in media containing 1% BSA plus the 0.1% fat emulsion. The results showed that only when the fat was present, LPL could reverse the function of ZHX2 on HepG2 cells.4. LPL reverses the growth inhibition of H22 tumor by ZHX2 in vivo.We injected 5×10∧6 H22 cells to BALB/C mice subcutaneously. Then injected these plasmids into tumor every other day:pcDNA3.0, pcDNA3.0-ZHX2-HA, pcDNA3.0-LPL-HA, ZHX2 plus LPL. The results showed that LPL promoted the growth of tumor, and could reverse the inhibition function of ZHX2 on tumor.Taken together, we clarify that ZHX2 represses LPL-mediated lipolysis and uptake of lipid, in doing so, protects the liver from NAFLD and HCC.Part II ZHX2 enhances the chemotherapy sensitivity of tumor cells by down-regulating the expression of MDR1Overexpression of multidrug resistance protein 1(MDR1, also known as p-glycoprotein), an ATP-dependent pump, causes the efflux of various hydrophobic compounds and xenobiotics leading to MDR. Previous studies have identified NF-Y as a central mediator of MDR1 activation.We previously reported the tumor suppressor function of Zinc-fingers and homeoboxes 2 (ZHX2) in hepatocellular carcinoma (HCC). Other studies indicate the association of increased ZHX2 expression with improved response to high dose chemotherapy in multiple myeloma. Based on the function of ZHX2 as a transcriptional repressor and its known interaction with NF-Y A, we hypothesized that ZHX2 might inhibit MDR1 expression in cancer cells, resulting in reduced efflux of chemotherapeutic drugs and subsequent increased sensitivity to these agents. Here, we aim to test whether increased ZHX2 levels in tumor cells repress multidrug resistance 1(MDR1) expression resulting in increased sensitivity to chemotherapeutic drugs.Ⅰ.ZHX2 enhances the chemotherapy sensitivity of various tumor cell lines. 1. ZHX2 expression level correlated with chemotherapy sensitivity in various cell lines. First of all, ZHX2 mRNA levels were compared in several tumor cell lines. Interestingly,ZHX2 expression level correlated with CDDP sensitivity in these cell lines. 2. ZHX2 enhances the chemotherapy sensitivity of HCC and lung cancer cells to CDDP and ADM. To explore further the relationship between ZHX2 and chemotherapy sensitivity, ZHX2 was overexpressed or knocked down by transient transfection. After 24 hours, cells were treated with CDDP or ADM and cultured for another 24 hours. The IC50 was calculated finally. In ZHX2-overexpressing cell lines (HepG2,HepG2.2.15 and SPC-A-1), the IC50 decreased significantly after treatment with both CDDP and ADM compared to pcDNA3.0-transfected cells treated with these drugs. In accordance, knock-down of ZHX2 in SMMC7721 and A549 cells increased the IC50 of both CDDP and ADM. These data indicate that increased ZHX2 levels result in increased sensitivity of tumor cells to these chemotherapeutic drugs.3. ZHX2 promotes the apoptosis of HepG2 cells induced by CDDP.HepG2 cells transfected with pcDNA3.0 or pcZHX2 were treated with CDDP for 24 hours. Flow cytometry after staining with PI and Annexin V to determine the percentage of Annexin V-stained cells. The results showed that increased ZHX2 levels in HepG2 cells enhance CDDP-induced apoptosis. Ⅱ.ZHX2 negatively regulates the expression of MDR1 in various tumor cell lines. To explore further the relationship between ZHX2 and MDR1, ZHX2 was overexpressed or knocked down by transient transfection. ZHX2 overexpression led to decreased MDR1 mRNA levels in HepG2, HepG2.2.15 and SPC-A-1 cells, whereas ZHX2 knockdown with two different siRNAs (ZHX2-1674, ZHX2-2360) resulted in elevated MDR1 mRNA levels in SMMC7721 and A549 cells. This difference was also seen at the protein level as determined by western blot. These data support the possibility that ZHX2 represses MDR1 expression in tumor cells.Ⅲ. ZHX2 reduces drug efflux from tumor cellsMDR1 is a well-known ATP-dependent drug efflux pump. To evaluate the effect of ZHX2 on regulating the MDR1 transporter activity, HepG2 and SPC-A-1 cells were transfected with pEGFP-ZHX2 and then treated with ADM, which emits a natural red fluorescence. EGFP-ZHX2 expression and ADM autofluorescence intensity were detected by fluorescence microscopy. Greater ADM accumulation in EGFP-ZHX2 transfected cells was dedeted.Enhanced ADM accumulation in EGFP-ZHX2 expressing HepG2 cells was further confirmed by flow cytometry. The red MFI in EGFP-positive cells was significantly higher than that in EGFP-negative cells 4 hours after ADM treatment. The red MFI in EGFP-positive cells remained higher than EGFP-negative cells 2 hours after ADM withdraw, suggesting enhanced ADM retention in EGFP-ZHX2 overexpressing cells. Consistently, EGFP-ZHX2 positive cells exhibited a decreased ADM releasing index compared with EGFP-ZHX2 negative cells. Taken together, these data suggest that ZHX2 suppresses MDR1 expression and decreases drug efflux, resulting in increased intracellular ADM levels. Ⅳ.ZHX2 down-regulates the expression of MDR1 by interacting with NF-YA1. ZHX2 inhibits the activity of MDR1 promoter in a dose-dependent manner.Previous studies indicate that ZHX2 functions as a transcriptional repressor. We therefore tested whether ZHX2 represses MDR1 promoter activity. SMMC7721 and A549 cells were transfected with pGL3-Mp and siCON or ZHX2 siRNAs or with pGL3-Mp and increasing amounts of ZHX2 siRNAs. Results of cotransfection and luciferase assays showed that ZHX2 knock-down by siRNAs greatly enhanced the MDR1 promoter activity in SMMC7721 and A549 cells. The increase in luciferase levels showed a dose-dependent response.1. ZHX2 inhibits the activity of MDR1 promoter in a NF-YA-dependent manner.The pGL3-MDR1-mutant (pGL3-mMp) was constructed. The results of cotransfection and luciferase reporter assays showed that ZHX2 could not repress the activity of the mutant plasmid. In order to identify whether NF-YA plays a role in the regulation of MDR1 by ZHX2, we cotransfected the NF-YA siRNA and pcDNA3.0-ZHX2-HA with MDR1 promoter. The results suggested that ZHX2 could not inhibit the activity of MDR1 promoter when co-transfection with NF-YA siRNA2. ZHX2 combines with NF-YA confirmed by Co-IP.HepG2 cells were transfected with pcDNA3.0-ZHX2-HA and then extracted the total protein. The result of immunoprecipitation showed that NF-YA protein could bind to ZHX2.3. ZHX2 interferes with the binding of NF-YA to MDR1 promoter. To directly test whether ZHX2 bound to the MDR1 promoter, ChIP were performed using HepG2 and SPC-A-1 cells transfected with pcZHX2-HA or pcEGFP-HA and oligonucleotides specific for the MDR1 promoter. As results shown, a specific PCR amplification was detected in the anti-HA immunoprecipitation from tumor cells transfected with pcZHX2-HA but not in pcEGFP-HA, indicating that ZHX2 bound to the MDR1 promoter. Interestingly, a relative weak PCR amplification was detected in the anti-NF-YA immunoprecipitation from tumor cells transfected with pcZHX2-HA than that in pcEGFP-HA, suggested that the presence of transfected ZHX2 could influence the NF-YA binding to the MDR1 promoter.HEK293 cells were transfected with pcDNA3.0, pcDNA-NF-YA, or pcNF-YA plus pcZHX2 (1 ug,2ug) respectively, and then extracted the nuclear protein. The result of electrophoretic mobility shift assay (EMSA) showed that, ZHX2 nuclear protein could bind to the NF-YA probe. Additionally, the supershift line confirmed that ZHX2 could specifically bind to Octamer domain.Taken together, these data suggest that ZHX2 interacts directly with NF-Y binding site on the MDR1 promoter and that this interaction inhibits NF-Y-mediated activation of MDR1 transcription. Coincidence with that, the result of EMSA also showed binding of NF-YA probe and ZHX2 protein.V.The expression of ZHX2 in clinical tumor samples is negative correlated with MDR1 expresssionIn order to evaluated the correlation of ZHX2 and MDR1 expression in tumor tissues,30 HCC samples and 54 lung cancer (33 lung squamous carcinomas,21 lung adenocarcinomas) samples were involved to do immunohistochemical staining with antibodies against ZHX2 and MDR1. Analysis results of Chi-square test and non-parametric test further confirmed the reverse correlation of nucleic ZHX2 with MDR1 in HCC and lung cancer. These indicated that reduced nuclear ZHX2 level might be responsible for enhanced MDR1 expression in tumor tissue.Taken together, we clarify that ZHX2 represses NF-Y-mediated activation of MDR1 transcription and, in doing so, enhances the effects of chemotherapeutics in tumor cells.